Combination therapy

ABSTRACT

The invention relates to pharmaceutical compositions comprising: (a) at least one angiotensin receptor blocker or a pharmaceutically acceptable salt thereof, and (b) at least one chemokine receptor pathway inhibitor or a pharmaceutically acceptable salt thereof. The invention also relates to pharmaceutical compositions comprising: (a) at least one angiotensin receptor blocker or a pharmaceutically acceptable salt thereof; and (b) at least one chemokine receptor pathway inhibitor or a pharmaceutically acceptable salt thereof which inhibits a component of the chemokine receptor pathway other than the chemokine receptor. Oral sustained release pharmaceutical compositions comprising the pharmaceutical composition, as well as injectable sustained release pharmaceutical compositions comprising the pharmaceutical composition are described. The invention further relates to tablets, capsules, injectable suspensions, and compositions for pulmonary or nasal delivery comprising the pharmaceutical composition. Also described are: methods for assessing the efficacy of the pharmaceutical composition; methods for assessing the inhibition or partial inhibition activity of the pharmaceutical composition; methods for the treatment, amelioration or prevention of a condition or disease comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition; and the use of the pharmaceutical composition for the manufacture of a dosage form for the treatment of a disease.

FIELD OF THE INVENTION

The present invention relates to a combination therapy, comprising atleast one chemokine receptor pathway inhibitor and at least oneangiotensin receptor blocker.

BACKGROUND ART

Proteins do not act in isolation in a cell, but in stable or transitorycomplexes, with protein-protein interactions being key determinants ofprotein function (Auerbach et al., (2002), Proteomics 2:611-623).Furthermore, proteins and protein complexes interact with other cellularcomponents like DNA, RNA and small molecules. Understanding both theindividual proteins involved in these interactions and theirinteractions are important for a better understanding of biologicalprocesses.

The primary physiological function of chemokines reported by Allen(Allen, S. et al. (2007) Annual Review Immunology 25:787-820) is theregulation of “cell migration during routine immune surveillance,inflammation and development”. Chemokines are released in response toproinflammatory cytokines and selectively bind to a large family of Gprotein-coupled receptors, which mediate the physiological responses tochemokines. Chemokines were originally referred to as chemotacticcytokines.

Since discovering that the chemokine system plays an integral role inhuman immunodeficiency virus (HIV) infection and the pathogenesis ofacquired immune deficiency syndrome (AIDS), considerable efforts havebeen made to understand the underlying mechanism(s) involved in order todevelop potential intervention strategies (Lusso, P. (2006) EMBO Journal25:447-456). Furthermore, any deleterious immune response associatedwith a particular condition, including asthma, almost invariably resultfrom a dysfunctional chemokine system. The pathogenesis ofatherosclerosis has also been shown to involve chemokine signalingpathways, with the infiltration of macrophages into arterial lesionsdirectly contributing to this aberrant inflammatory disorder (Boisvert,W. (2004) Trends in Cardiovascular Medicine 14:161-165).

Animal model studies of chronic inflammatory diseases have demonstratedthat inhibition of binding between MCP-1 (monocyte chemotacticprotein-1, also known as monocyte chemoattractant protein-1, monocytechemotactic and activating factor (MCAF) and chemokine (C-C motif)ligand 2 (CCL2)) and CCR2 (chemokine (C-C motif) receptor 2) by anantagonist suppresses the inflammatory response. The interaction betweenMCP-1 and CCR2 has been implicated (see Rollins B J (1996) Mol. Med.Today, 2:198; and Dawson J, et al., (2003) Expert Opin. Ther. Targets,7(1):35-48) in inflammatory disease pathologies such as uveitis,atherosclerosis, rheumatoid arthritis, multiple sclerosis, Crohn'sDisease, nephritis, organ allograft rejection, fibroid lung, renalinsufficiency, diabetes and diabetic complications, diabeticnephropathy, diabetic retinopathy, diabetic retinitis, diabeticmicroangiopathy, tuberculosis, sarcoidosis, invasive staphylococcia,inflammation after cataract surgery, allergic rhinitis, allergicconjunctivitis, chronic urticaria, allergic asthma, periodontaldiseases, periodonitis, gingivitis, gum disease, diastoliccardiomyopathies, cardiac infarction, myocarditis, chronic heartfailure, angiostenosis, restenosis, reperfusion disorders,glomerulonephritis, solid tumors and cancers, chronic lymphocyticleukemia, chronic myelocytic leukemia, multiple myelorna, malignantmyeloma, Hodgkin's disease, and carcinomas of the bladder, breast,cervix, colon, lung, prostate, or stomach.

Monocyte migration is inhibited by MCP-1 antagonists (either antibodiesor soluble, inactive fragments of MCP-1), which have been shown toinhibit the development of arthritis, asthma, and uveitis.Propagermanium (3-oxygermylpropionic acid polymer), a molecule that hasbeen used as a therapeutic agent against chronic hepatitis, also hasbeen shown to specifically inhibit in vitro chemotactic migration ofmonocytes by MCP-1 through a mechanism that seems to requireglycosylphosphatidylinositol (GPI)-anchored proteins such as CD 55, CD59and CD16 (Yokochi, S. (2001) Journal of Interferon and Cytokine Research21:389-398).

Both MCP-1 and CCR2 knockout (KO) mice have demonstrated that monocyteinfiltration into inflammatory lesions is significantly decreased. Inaddition, such KO mice are resistant to the development of experimentalallergic encephalomyelitis (EAE, a model of human MS), cockroachallergen-induced asthma, atherosclerosis, and uveitis. Rheumatoidarthritis and Crohn's Disease patients have improved during treatmentwith TNF-α antagonists (e.g., monoclonal antibodies and solublereceptors) at dose levels correlated with decreases in MCP-1 expressionand the number of infiltrating macrophages.

MCP-1 has been implicated in the pathogenesis of seasonal and chronicallergic rhinitis, having been found in the nasal mucosa of mostpatients with dust mite allergies. MCP-1 has also been found to inducehistamine release from basophils in vitro. During allergic conditions,both allergens and histamines have been shown to trigger (i.e., toup-regulate) the expression of MCP-1 and other chemokines in the nasalmucosa of people with allergic rhinitis, suggesting the presence of apositive feedback loop in such patients.

Kidney disease is associated with chronic inflammation characterised bythe accumulation of kidney macrophages. The production of monocytechemoattractant protein-1 (MCP-1/CCL2) by diabetic kidneys has beenidentified as a major factor influencing macrophage accumulation in thekidney disease arising from diabetic nephropathy (see Tesch G H (2008)MCP-1/CCL2: a new diagnostic marker and therapeutic target forprogressive renal injury in diabetic nephropathy Am J Physiol RenalPhysiol 294:697-701). In various animal models inhibition of CCR2 and/orinhibition of specific CCR2 pathways and/or inhibition of the CCR2ligand MCP-1 has been shown to reduce kidney damage (see Tesch (2008)above; Rao V et al (2006) Role for Macrophage Metalloelastase inGlomerular Basement Membrane Damage Associated with Alport Syndrome,American Journal of Pathology, Vol. 169(1) 32-46; Kang Y S (2010) CCR2antagonism improves insulin resistance, lipid metabolism, and diabeticnephropathy in type 2 diabetic mice Kidney International 78, 883-894;Kitagawa K (2004) Blockade of CCR2 Ameliorates Progressive Fibrosis inKidney, American Journal of Pathology, Vol. 165(1) 237-246; Park J(2008) MCP-1/CCR2 system is involved in high glucose-induced fibronectinand type IV collagen expression in cultured mesangial cells, Am JPhysiol Renal Physiol 295: F749-F757).

Tesch (2008) notes that selective targeting of MCP-1 has been proven tobe an effective treatment in suppressing animal models of kidney diseasethat include diabetic nephropathy; however, such therapies have not yetbeen validated in human diabetic nephropathy. Treatments including smallmolecular antagonists of CCR2 (INCB3344, propagermanium, RS-504393) havebeen shown to suppress inflammation in mouse models of multiplesclerosis, renal ischemia-reperfusion injury, ureter obstruction, anddiabetic nephropathy and in a rat model of arthritis; Engineeredbiological antagonists of CCR2 have also proven effective; Subcutaneousinfusion of cells transfected with a vector expressing a truncatedinactive form of MCP-1 has been found to suppress the development ofrenal inflammation in a mouse model of lupus nephritis. Similarly,muscle transfection with 7ND (a mutant of MCP-1) reduces renalinflammation in mouse models of renal ischemia-reperfusion injury, lupusnephritis, and diabetic nephropathy. Human trials of chemokinemonotherapies for inflammatory diseases, to date, have not lead to drugapprovals. Anders A-J et al considers reasons why single chemokineantagonist treatments have not been effective in disease treatments anddiscuss possible explanations including redundancy of single chemokinemediators and variable expression patterns of chemokine receptors. (seeAnders A-J et al (2009) Questions about Chemokine and Chemokine ReceptorAntagonism in Renal Inflammation, Nephron Exp Nephrol 2010;114:e33-e38). Therefore, there exists a need in the art for an effectivetreatment of diseases that are caused through the CCR2 pathways.

The renin-angiotensin system (RAS) plays an important role in thesympathetic nervous system and fluid homeostasis. Renin is a proteolyticenzyme secreted by the kidneys that mediates the formation ofangiotensin I (AngI) from a globulin precursor, angiotensinogen (Rang,H. P., et al., Pharmacology: 3^(rd) Edition, 1995, Published byChurchill Livingstone, Edinburgh, UK.). AngI itself appears to havelittle physiological importance other than providing a substrate for asecond enzyme, angiotensin-converting enzyme (ACE), which converts AngIto the highly active angiotensin II (AngII). However, it should be notedthat AngII can be generated by alternative, ACE-independent mechanisms.AngII can in turn be metabolised to AngIII by aminopeptidases.

AngII is an extremely potent vasoconstrictor and as a consequence it hasbeen extensively studied in the context of heart disease andhypertension pathogenesis (Ramasubbu, K. (2007) Cardiology Clinics25:573-580).

Chronic renal disease is a major cause of mortality and morbidity,however the basic cellular events that promote its progression remainelusive and it is likely that proinflammatory mediators, leading toinflammation, hypoxia and increased extracellular matrix (ECM)deposition are a major cause of renal failure (Gilbert (1999) Kidney Int56:1627-1637, 1999). These pathological events are accompanied byproteinuria and a decline in glomerular filtration rate (GFR),ultimately leading to end-staged renal failure. Althoughangiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptorblockers are now viewed as first line treatment for chronic renaldisease and have clearly been shown to confer renoprotection, chronicrenal disease remains a progressive disorder, which ultimately leads torenal failure. In the collaborative study group trial (Lewis (1993). Theeffect of angiotensin converting enzyme inhibition on diabeticnephropathy. New England Journal of Medicine 329:1456-1462), captopriltherapy, although retarding the decline in renal failure, did not haltthe progression of diabetic nephropathy in the vast majority ofpatients. In contrast to the clinical setting, experimental studies ofrenoprotective agents have mostly shown complete amelioration of renalstructural and functional abnormalities in commonly used models ofdiabetic nephropathy. A major advantage of the diabetic Ren-2 rat andthe Sub Total nephrectomy (STNx) model is that as is observed in man,ACEi or angiotensin receptor blockers attenuate but do not prevent thedevelopment of renal failure (Kelly (1998) Kidney Int 54:343-352, andKelly (2000) Kidney Int 57:1882-1894). Furthermore, the diabetic Ren-2rat and STNx models can be used to study additional therapies which havethe potential to further improve the outlook in renal diseaseprogression in the context of concomitant ACEi or angiotensin receptorblockade.

The renin-angiotensin system (RAS), a hormonal cascade involved in bloodpressure control, electrolyte homeostasis and cell growth and death,exists in the kidney at two major sites: the glomerulus and proximaltubules. The RAS has been implicated in the progression of kidneydisease as blockade of this system attenuates proteinuria and glomerularand tubulointerstitial disease in both human and experimental diabetesLewis (1993). The renoprotective effect of RAS blockers have beenattributed to their ability to reduce glomerular pressure (Zatz (1985)Predominance of hemodynamic rather than metabolic factors in thepathogenesis of diabetic nephropathy. PNAS 82:5963-5967). However it hasbeen recognized that local increases in angiotensin II can inducesclerosis and inflammation through its cell growth promoting properties(Wolf (1993) Angiotensin II as a renal growth factor. J Am Soc Nephrol3:1531-1540). There is ample evidence from studies of various glomerulardiseases that Ang II exerts cell injury by the up-regulation of othergrowth factors (Ruiz Ortega (1994) Involvement of angiotensin II andendothelin in matrix protein production and renal sclerosis. J HypertensSuppl. 12:S51-S58) such as transforming growth factor-(TGF-β). Indeed,these growth factors are produced by the kidney and are increased by AngII, inducing cell proliferation, cell cycle arrest, and death,alterations in cell phenotype and ECM accumulation (Kelly (1998), Kelly(2000) and Kelly (2002)). Although evidence suggests that Ang II inducesa variety of responses by the upregulation of growth factors, very fewstudies have described how Ang II promotes activation of the growthfactors in the diabetic setting (Naito (2004) Am J Physiol Renal Physiol286:F278-F287).

The efficacy of the angiotensin receptor blocker irbesartan (AT₁R,market name Avapro®, SanofiAventis) in the management of diabeticnephropathy in patients with hypertension has been evaluated in twolarge (n>500), randomized, double-blind, placebo-controlled,multinational trials, IRMA 2 (Irbesartan Microalbuminuria Type 2Diabetes in Hypertensive Patients) (Parving (2001) N Engl J Med 345(12): 870-8 and IDNT (Irbesartan Diabetic Nephropathy Trial) Lewis(2001) N Engl J Med 345 (12): 851-60).

In order to counter the deleterious vasoconstrictor effects of AngII inpatients with hypertension [onset of end stage renal disease],therapeutic strategies have been developed that intervene at the levelof AngII signalling. In particular, compounds that inhibit the activityof ACE, preventing the conversion of AngI to AngII, and those thatspecifically block the activation of angiotensin receptors (ATRs), havebeen employed in the treatment of such conditions (Matchar, D. B. (2008)Annals of Internal Medicine 148:16-29).

The inventors have shown the heteromerisation of the angiotensinreceptor with members of the chemokine receptor family (WO2010/108232).The inventors have shown that the chemokine receptor associates with theangiotensin receptor as a chemokine receptor/angiotensin receptorhetero-dimer/-oligomer. The inventors have shown that the CCR2associates with the AT1R as a CCR2/AT1R hetero-dimer/-oligomer.

Angiotensin and CCR2 signalling pathways have previously been shown tointeract. For example: angiotensin II effects on vascular pathologiesare attenuated by deficiency of the CCR2 receptor (Daugherty A (2010)Clin Sci (Lond). 118(11):681-9; Ishibachi M (2004) Arteriosclerosis,Thrombosis, and Vascular Biology 24; Tieu (2011) Aortic AdventitialFibroblasts Participate in Angiotensin-Induced Vascular WallInflammation and Remodelling J Vasc Res 48(3) 261-272).

Furthermore, angiotensin II, which induces MCP-1 expression, increasewith age resulting in upregulation of MCP-1 and its receptor CCR2. Thisupregulation can also occur in various diseases. (Spinetti G (2004)Arterioscler Thromb Vasc Biol 24(8): 1397-402).

Angiotensin receptor blockers have been shown to inhibit the expressionof MCP-1 and CCR2 (Dai (2007) British Journal of Pharmacology 152,1042-1048).

The inventors have surprisingly found that the administration of anangiotensin receptor blocker together with a chemokine receptor pathwayinhibitor overcomes some or all of the shortcomings of the prior art.

The preceding discussion is intended only to facilitate an understandingof the invention. It should not be construed as in any way limiting thescope or application of the following description of the invention, norshould it be construed as an admission that any of the informationdiscussed was within the common general knowledge of the person skilledin the appropriate art at the priority date.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition comprising:

-   -   a) at least one angiotensin receptor blocker or a        pharmaceutically acceptable salt thereof, and    -   b) at least one chemokine receptor pathway inhibitor or a        pharmaceutically acceptable salt thereof.

The pharmaceutical composition may optionally further comprise apharmaceutically acceptable carrier.

In one aspect, the pharmaceutical composition inhibits or partiallyinhibits arrestin recruitment.

In another aspect, the pharmaceutical composition inhibits or partiallyinhibits inositol phosphate production.

The invention further provides a method for the treatment, ameliorationor prevention of a condition or disease comprising administering to asubject a therapeutically effective amount of a combination of (i) anangiotensin receptor blocker and (ii) a chemokine receptor pathwayinhibitor.

In one aspect, the chemokine receptor pathway inhibitor inhibits orpartially inhibits a protein other than the chemokine receptor, morepreferably, the inhibitor is an agent which blocks MCP-1 inducedmigration and activation of monocytes and chemotactic migration throughthe targeting of glycosylphosphatidylinositol (GPI)-anchored proteinssuch as CD55, CD59 and CD16. Preferably, the chemokine receptor pathwayinhibitor is propagermanium. Alternatively, the chemokine receptorpathway inhibitor is RS504393.

Preferably, the angiotensin receptor blocker is irbesartan.

The invention also contemplates the use of a pharmaceutical compositioncomprising at least one angiotensin receptor blocker or apharmaceutically acceptable salt thereof, and at least one chemokinereceptor pathway inhibitor or a pharmaceutically acceptable saltthereof; for the manufacture of a dosage form for the treatment of adisease.

The pharmaceutical composition may optionally further comprise apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows a bar graph of the improvement in levels of proteinureaachieved in the sub-total nephrectomy (STNx) model of end organ renaldisease when treated with a combination of Irbesartan (Irb), anangiotensin receptor blocker, and propagermanium (PPG) (a chemokinereceptor pathway inhibitor) (low dose) as compared to untreated animalsand animals treated with the angiotensin receptor blocker alone asdescribed in Example 1.

FIG. 2: shows a bar graph of the improvement in levels of proteinureaachieved in the sub-total nephrectomy (STNx) model of end organ renaldisease when treated with a combination of Irbesartan (Irb), anangiotensin receptor blocker, and propagermanium (PPG) (a chemokinereceptor pathway inhibitor) (high dose) as compared to untreatedanimals, animals treated with PPG alone, and animals treated with theangiotensin receptor blocker alone as described in Example 2.

FIG. 3: shows representative phase-contrast images of histologicalsections obtained from the sub-total nephrectomy (STNx) model of endorgan renal disease described in Example 1 showing renal samplesobtained from untreated or control animals (A); STNx only (B); STNx andpropagermanium (C); STNx and Irbesartan (D); and STNx and Irbesartan incombination (E). Intense brown stained cells in glomerulus representpodocytes.

FIG. 4: shows a bar graph of the improvement in numbers of podocytesachieved in the sub-total nephrectomy (STNx) model of end organ renaldisease when treated with a combination of Irbesartan (Irb), anangiotensin receptor blocker, and propagermanium (PPG) (a chemokinereceptor pathway inhibitor) (high dose) as compared to untreatedanimals, animals treated with PPG alone, and animals treated withIrbesartan (the angiotensin receptor blocker) alone as described inExample 2.

FIG. 5: shows bar graphs indicating the effect of AT1R and CCR2 blockadeon β-arrestin2 recruitment as measured by Ligand-induced BRET anddescribed in Example 3. HEK293FT cells were transiently transfected bythe plasmids coding for β-arrestin2-Venus and the indicated receptors:CCR2-Rluc8 (top panel), AT1R-Rluc8 (middle panel) or AT1R-Rluc8 and theuntagged CCR2 (bottom panel). 48 h post-transfection, cells were used togenerate the agonist-induced BRET signal in live cells. For this, cellswere first pre-incubated or not for 30 minutes at 37° C. with Irbesartan(10 μM), RS504393 (10 μM) or both combined. Then cells were stimulatedor not for 30 minutes at 37° C. with 100 nM of AngII, MCP-1 or bothtogether and the BRET signal was measured. Data represent mean±SEM ofthree independent experiments performed in triplicate.

FIG. 6: shows bar graphs indicating effect of AT1R and CCR2 blockade oninositol phosphate production as described in Example 4. HEK293FT cellswere transiently transfected with the plasmids coding for AT1R-Rluc8 andβ-arrestin2-Venus in the absence (top panel) and presence (bottom panel)of untagged CCR2. 48 h post-transfection, cells were used to generatethe agonist-induced inositol (1) phosphate (IP1) productionmeasurements. For this, cells were first pre-incubated or not for 30minutes at 37° C. with Irbesartan (10 μM), RS504393 (10 μM) or bothcombined. Then cells were stimulated or not for 30 minutes at 37° C.with 100 nM of AngII, MCP-1 or both together and IP1 production wasmeasured. Data are normalized as a percentage of AngII-induced IP1production in cells expressing AT1R alone. Data represent mean±SEM ofthree independent experiments performed in triplicate.

FIG. 7: shows bar graphs indicating the effect of AT1R and CCR2 blockadeon β-arrestin2 recruitment as measured by Ligand-induced BRET anddescribed in Example 5. HEK293FT cells were transiently transfected bythe plasmids coding for CCR2-Rluc8 and β-arrestin2-Venus in the absence(top panel) and presence (bottom panel) of haemagglutinin (HA)-taggedAT1R. 48 h post-transfection, cells were used to generate theagonist-induced BRET signal in live cells. For this, cells were firstpre-incubated or not for 30 minutes at 37° C. with Irbesartan (10 μM),EXP3174 (the active metabolite of Losartan; μM), RS504393 (10 μM) orcombinations of Irbesartan and RS504393 or EXP3174 and RS504393. Thencells were stimulated or not for 30 minutes at 37° C. with 100 nM ofAngII, MCP-1 or both together and the BRET signal was measured.

FIG. 8: shows bar graphs indicating effect of AT1R and CCR2 blockade oninositol phosphate production as described in Example 6. HEK293FT cellswere transiently transfected with the plasmids coding for AT1R-Rluc8 andβ-arrestin2-Venus in the absence (top panel) and presence (bottom panel)of untagged CCR2. 48 h post-transfection, cells were used to generatethe agonist-induced inositol (1) phosphate (IP1) productionmeasurements. For this, cells were first pre-incubated or not for 30minutes at 37° C. with EXP3174 (the active metabolite of Losartan; 10μM), RS504393 (10 μM) or both combined. Then cells were stimulated ornot for 30 minutes at 37° C. with 100 nM of AngII, MCP-1 or bothtogether and IP1 production was measured. Data are shown as induced IP1(arbitrary units).

FIG. 9: shows dose-response curves indicating the effect of activatingCCR2 in the absence and presence of activated AT1R in terms of Gα_(i1)coupling as measured by Ligand-induced BRET and described in Example 7.HEK293FT cells were transiently transfected by the plasmids coding forGαi1-Rluc8 and CCR2-YFP in the absence (top panel) and presence (bottompanel) of haemagglutinin (HA)-tagged AT1R. 48 h post-transfection, cellswere used to generate the agonist-induced BRET signal data in live cellsat various concentrations of MCP-1 or at various concentrations of AngIIin the presence of 100 nM MCP-1.

ABBREVIATIONS

-   ACE Angiotensin-converting enzyme-   ACEi Angiotensin-converting enzyme inhibitor-   AIDS Acquired immune deficiency syndrome-   AngI Angiotensin I peptide-   AngII Angiotensin II peptide-   AngIII Angiotensin III peptide-   AT₁R Angiotensin receptor type 1-   AT₂R Angiotensin receptor type 2-   barr beta-arrestin.-   BP Blood pressure-   CCL2 Chemokine (C-C motif) ligand 2-   CCRs CC Chemokine receptors-   DOP Delta opioid-   GFR Glomerular filtration rate-   GPCRs G protein-coupled receptors.-   HIV Human immunodeficiency virus-   KOP Kappa opioid-   LPO Lateral preoptic area-   MCP-1 Monocyte chemotactic protein-1, also known as monocyte    chemoattractant protein-1-   NPY Neuropeptide Y.-   STNx Sub-total nephrectomy

DESCRIPTION OF THE INVENTION

General

All publications, including patents and patent applications, citedherein, whether supra or infra, are hereby incorporated by reference intheir entirety. However, publications mentioned herein are cited for thepurpose of describing and disclosing the protocols, reagents and vectorsthat are reported in the publications and which may be used inconnection with the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include the plural unless the context clearly dictatesotherwise. Thus, for example, a reference to “a protein” includes aplurality of such proteins, and a reference to “an analyte” is areference to one or more analytes, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any materials andmethods similar or equivalent to those described herein can be used topractice or test the present invention, the preferred materials andmethods are now described.

The invention described herein may include one or more ranges of values(e.g. size, concentration etc). A range of values will be understood toinclude all values within the range, including the values defining therange, and values adjacent to the range that lead to the same orsubstantially the same outcome as the values immediately adjacent tothat value which defines the boundary to the range.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations, such as “comprises” or “comprising”will be understood to imply the inclusion of a stated integer, or groupof integers, but not the exclusion of any other integers or group ofintegers.

DETAILED DESCRIPTION OF THE INVENTION

Recent studies have shown that G protein-coupled receptors (GPCRs) maynot only act as monomers but also as homo- and hetero-dimers and/orhomo- and hetero-oligomers (also known as homomers and heteromers),which causes altered ligand binding, signalling and endocytosis (Rios etal. (2000) Pharmacol. Ther. 92:71-87). The effect of drugs acting asagonists or antagonists of a specific receptor may therefore depend onthe binding partners of this receptor. It may be desirable to limit theeffect of a drug to a cellular response mediated by a specific receptordimer or oligomer.

Instances of different tissues having different repertoires ofhetero-dimers have been reported. For example, 6′guanidinoaltrindole, ananalogue of a well-known kappa opioid (KOP) receptor ligand, has beenidentified as a delta opioid-kappa opioid (DOP-KOP) hetero-dimerselective agonist, with efficacy as a spinally selective analgesic,leading to the conclusion that DOP-KOP heterodimers are expressed in thespinal cord, but not in the brain (Waldhoer, M. et al. (2005) Proc.Natl. Acad. Sci. USA 102:9050-9055). Accordingly, the hetero-dimeric orhetero-oligomeric receptor, comprising at least one chemokine receptorsubunit associated with at least one angiotensin receptor subunitrepresents a novel drug target.

As is the case with 6′guanidinoaltrindole, known ligands may exhibitdiffering abilities to trigger a hetero-dimeric receptor, which mayuncover new applications for pre-existing molecules:

-   -   Hilairet S. et al. 2003 (J. Biol. Chem. 278:23731-23737) have        recently shown that CB1 antagonists suppress appetite by acting        through a CB1/OxR1 heteromer pair.    -   It has been shown that somatostatin SSTR5 receptor will        heteromerise with a dopamine D2 receptor (Rocheville M. et        al. (2000) Science 288:154-157).    -   Angiotensin and CCR2 signalling pathways have previously been        shown to interact. For example, angiotensin II effects on        vascular pathologies are attenuated by deficiency of the CCR2        receptor (Daugherty A (2010) Clin Sci (Lond). 118(11):681-9;        Ishibachi M (2004) Arteriosclerosis, Thrombosis, and Vascular        Biology 24).    -   The inventors have shown the heteromerisation of the angiotensin        receptor with members of the chemokine receptor family        (WO2010/108232).

Whilst these examples show the functional interaction of receptors, theydo not identify the specific formulations of combined therapies that mayprovide improved therapeutic outcomes.

Pharmaceutical Compositions

Preferably, the combination therapy will act through the chemokinereceptor pathway and the angiotensin receptor. The present inventiontherefore provides a pharmaceutical composition comprising:

-   -   a) at least one angiotensin receptor blocker or a        pharmaceutically acceptable salt thereof; and    -   b) at least one chemokine receptor pathway inhibitor or a        pharmaceutically acceptable salt thereof.

The pharmaceutical composition may optionally further comprise apharmaceutically acceptable carrier.

The phrase “chemokine receptor” is to be understood to at least includethe G protein-coupled CC chemokine receptors (CCRs), including: CCchemokine receptor 1 (CCR1), CC chemokine receptor 2 (CCR2), CCchemokine receptor 3 (CCR3), CC chemokine receptor 4 (CCR4), CCchemokine receptor 5 (CCR5), CC chemokine receptor 6 (CCR6), CCchemokine receptor 7 (CCR7), CC chemokine receptor 8 (CCR8), CCchemokine receptor 9 (CCR9), CC chemokine receptor 10 (CCR10). Thephrase “chemokine receptor” is also to be understood to include the Gprotein-coupled CXC chemokine receptors (CXCRs), including: CXCchemokine receptor 1 (CXCR1), CXC chemokine receptor 2 (CXCR2), CXCchemokine receptor 3 (CXCR3), CXC chemokine receptor 4 (CXCR4), CXCchemokine receptor 5 (CXCR5), CXC chemokine receptor 6 (CXCR6) and CXCchemokine receptor 7 (CXCR7). The phrase “chemokine receptor” is to befurther understood to include the G protein-coupled XC chemokinereceptor 1 (XCR1). The phrase “chemokine receptor” is to be furtherunderstood to include the G protein-coupled CX3 chemokine receptor(CX₃CR1). The phrase “chemokine receptor” is to be further understood toinclude the G protein-coupled CCX-CKR chemokine receptor (CCX-CKR). Thephrase “chemokine receptor” is to be further understood to include the Gprotein-coupled D6 chemokine receptor (D6). The phrase “chemokinereceptor” is to be further understood to include the G protein-coupledDARC/Duffy chemokine receptor (DARC). This list of chemokine receptorsis compiled from a review by Allen (Allen, S. et al. (2007) Chemokine:Receptor Structure, Interactions and Antagonism. Annual ReviewImmunology 25:787-820). Finally, the phrase “chemokine receptor” is tobe further understood to include any newly discoveredCCR/CXCR/XCR/CX₃CR/CCX-CKR/D6/DARC family members.

The chemokine receptor may be selected from the group comprising the CCchemokine receptor 1 (CCR1), CC chemokine receptor 2 (CCR2), CCchemokine receptor 3 (CCR3), CC chemokine receptor 4 (CCR4), CCchemokine receptor 5 (CCR5), CC chemokine receptor 6 (CCR6), CCchemokine receptor 7 (CCR7), CC chemokine receptor 8 (CCR8), CCchemokine receptor 9 (CCR9), CC chemokine receptor 10 (CCR10), CXCchemokine receptor 1 (CXCR1), CXC chemokine receptor 2 (CXCR2), CXCchemokine receptor 3 (CXCR3), CXC chemokine receptor 4 (CXCR4), CXCchemokine receptor 5 (CXCR5), CXC chemokine receptor 6 (CXCR6) and CXCchemokine receptor 7 (CXCR7), the G protein-coupled XC chemokinereceptor 1 (XCR1), the G protein-coupled CX3 chemokine receptor(CX₃CR1), the G protein-coupled CCX-CKR chemokine receptor (CCX-CKR),the G protein-coupled D6 chemokine receptor (D6), the G protein-coupledDARC/Duffy chemokine receptor (DARC), and aCCR/CXCR/XCR/CX₃CR/CCX-CKR/D6/DARC chemokine receptor.

The phrase “chemokine receptor pathway” is to be understood to at leastinclude any one of the pathways triggered by the chemokine receptorslisted above. Preferably, the chemokine receptor pathway is a pathwaytriggered by the G protein-coupled CC chemokine receptors (CCRs),including CC chemokine receptor 2 (CCR2).

The term “a component of the chemokine receptor pathway other than thechemokine receptor” as used herein, is to be understood as including acomponent of any one of the pathways listed above which is triggered byone or more of the chemokine receptors listed above, wherein thecomponent is itself not a chemokine receptor as listed above.Preferably, the component is a protein such as, but not limited to, atransduction or signalling protein. The component of the chemokinereceptor pathway may interact directly with the triggering chemokinereceptor. Alternatively, the component of the chemokine receptor pathwaymay interact indirectly with the triggering chemokine receptor by way ofprotein-protein interaction or complex formation. Alternatively, thecomponent of the chemokine receptor pathway may interact indirectly withthe triggering chemokine receptor by way of a signalling cascade such asis known in the art.

The phrase “chemokine receptor pathway inhibitor” is intended to includeany compound or agent which inhibits or partially inhibits any one ofthe pathways associated with the chemokine receptors listed above,including compounds or agents which inhibit components of the chemokinereceptor pathway other than the chemokine receptor itself. For example,the inhibitor may inhibit or partially inhibit proteins that associatewith chemokine receptors, or may inhibit compounds or pathway stepsbefore and/or after the chemokine receptor itself. Preferably, thechemokine receptor pathway inhibitor is a CCR2 antagonist, CCR2 inverseagonist or CCR2 negative allosteric modulator.

The chemokine receptor pathway inhibitor may be selected from the groupcomprising a direct CCR2 antagonist, an inverse CCR2 agonist, a negativeallosteric CCR2 modulator; an indirect CCR2 antagonist, an indirectinverse CCR2 agonist, and an indirect negative allosteric CCR2modulator.

More preferably the phrase includes any inhibitor which inhibits orpartially inhibits any one of the chemokine receptor pathways associatedwith MCP-1 and/or CCR2, which includes a direct CCR2 and/or MCP-1antagonist, inverse agonist or negative allosteric modulator; or anantagonist, inverse agonist or negative allosteric modulator which worksindirectly through blocking of these pathways at different levels.

More specifically, the phrase includes Propagermanium(3-oxygermylpropionic acid polymer), a molecule that has been used as atherapeutic agent against chronic hepatitis, also has been shown tospecifically inhibit in vitro chemotactic migration of monocytes byMCP-1 through a mechanism that seems to requireglycosylphosphatidylinositol (GPI)-anchored proteins such as CD 55, CD59and CD16 (Yokochi, S. (2001) Journal of Interferon and Cytokine Research21:389-398). Propagermanium is also known as3-[(2-Carboxyethyl-oxogermyl)oxy-oxogermyl]propanoic acid,proxigermanium, Ge-132, bis (2-carboxyethylgermanium) sesquioxide(CEGS), 2-carboxyethylgermasesquioxane, SK-818, organic germanium,germanium sesquioxide, 3,3′-(1,3-dioxo-1,3-digermanoxanediyl)bispropionic acid, 3-oxygermylpropionic acid polymer,poly-trans-(2-carboxyethyl) germasesquioxane, proxigermanium,repagermanium and Serocion. Propagermanium has the following formula:

The phrase also includes RS504393. RS504393 has the following formula:

The invention therefore also provides a pharmaceutical compositioncomprising:

-   -   a) at least one angiotensin receptor blocker or a        pharmaceutically acceptable salt thereof; and    -   b) at least one chemokine receptor pathway inhibitor or a        pharmaceutically acceptable salt thereof which inhibits a        component of the chemokine receptor pathway other than the        chemokine receptor.

The pharmaceutical composition may optionally further comprise apharmaceutically acceptable carrier.

In one preferred embodiment the chemokine receptor pathway inhibitor isselected from the group consisting of:

-   -   (i) antagonists of chemokine receptors or components of the        chemokine receptor pathway other than the chemokine receptor;    -   (ii) inverse agonists of chemokine receptors or components of        the chemokine receptor pathway other than the chemokine        receptor;    -   (iii) negative allosteric modulators of chemokine receptors or        components of the chemokine receptor pathway other than the        chemokine receptor;

A more specific example of a chemokine receptor pathway inhibitor whichtargets a component of the chemokine receptor pathway other than thechemokine receptor might be an agent which blocks pathways associatedwith MCP-1 induced migration, activation of monocytes and chemotacticmigration. Such agents that might be targeted includeglycosylphosphatidylinositol (GPI)-anchored proteins, and morespecifically CD55, CD59 and CD16.

Known antagonists of chemokine receptors include; RS504393, RS102895,MLN-1202 (Millennium Pharmaceuticals), INCB3344, INCB3284 and INCB8696(Incyte Pharmaceuticals), MK-0812 (Merck), CCX140 (ChemoCentryx),PF-4136309 (Pfizer), BMS-741672 (Bristol-Myers Squibb); Repertaxin(CXCR2), TAK-779 (CCR5), TAK-220 (CCR5), TAK-652 (CCR5), AK692 (CCR5),CMPD167 (CCR5), BX-471 (CCR1), AMD3100 (CXCR4), AMD11070 (CXCR4), FC131(CXCR4), MLN3897 (CCR1), CP-481715 (CCR1), GW-873140 (CCR5). Thechemokine receptor pathway inhibitor may be selected from the groupcomprising RS504393, RS102895, MLN-1202, INCB8696, MK-0812, CCX140,PF-4136309, BMS-741672; Repertaxin (CXCR2), TAK-779 (CCR5), TAK-220(CCR5), TAK-652 (CCR5), AK692 (CCR5), CMPD167 (CCR5), BX-471 (CCR1),AMD3100 (CXCR4), AMD11070 (CXCR4), FC131 (CXCR4), MLN3897 (CCR1),CP-481715 (CCR1), and GW-873140 (CCR5).

In one preferred embodiment the chemokine receptor pathway inhibitor isan antagonist of a chemokine receptor. In one preferred embodiment thechemokine receptor pathway inhibitor is selected from the groupconsisting of: RS504393, RS102895, MLN-1202 (MillenniumPharmaceuticals), INCB3344, INCB3284, INCB8696 (Incyte Pharmaceuticals),MK-0812 (Merck), CCX140 (ChemoCentryx), PF-4136309 (Pfizer), BMS-741672(Bristol-Myers Squibb); Repertaxin (CXCR2), TAK-779 (CCR5), TAK-220(CCR5), TAK-652 (CCR5), AK692 (CCR5), CMPD167 (CCR5), BX-471 (CCR1),AMD3100 (CXCR4), AMD11070 (CXCR4), FC131 (CXCR4), MLN3897 (CCR1),CP-481715 (CCR1) and GW-873140 (CCR5). In one preferred embodiment thechemokine receptor pathway inhibitor is not RS102895.

In one preferred embodiment the chemokine receptor pathway inhibitor ispropagermanium (also known as bis (2-carboxyethylgermanium) sesquioxide(CEGS), organic germanium, germanium sesquioxide,3,3′-(1,3-dioxo-1,3-digermanoxanediyl) bispropionic acid,3-oxygermylpropionic acid polymer, poly-trans-(2-carboxyethyl)germasesquioxane, proxigermanium, repagermanium and Serocion).

In one preferred embodiment the chemokine receptor pathway inhibitorinhibits the in vitro chemotactic migration of monocytes induced byMCP-1. In another preferred embodiment the chemokine receptor pathwayinhibitor inhibits the in vitro chemotactic migration of monocytesinduced by MCP-1 through a mechanism requiringglycosylphosphatidylinositol (GPI)-anchored proteins such as CD 55, CD59and CD16. In another preferred embodiment the chemokine receptor pathwayinhibitor stabilizes the complexes CCR2/CD55 and/or CCR2/CD59 and/orCCR2/CD16. The chemokine receptor pathway inhibitor may be a peptide,polypeptide or small chemical entity. For example, the chemokinereceptor pathway inhibitor may be a protein, binding protein orantibody.

The chemokine receptor pathway inhibitor may inhibit MCP-1 inducedmigration and activation of monocytes and chemotactic migration throughthe targeting of one or more glycosylphosphatidylinositol (GPI)-anchoredproteins selected from the group comprising CD55, CD59 and CD16. Thechemokine receptor pathway inhibitor may stabilize the complexesCCR2/CD55 and/or CCR2/CD59 and/or CCR2/CD16.

Propagermanium is a chemokine receptor pathway inhibitor, but it doesnot inhibit MCP-1 binding and appears to targetglycosylphosphatidylinositol (GPI)-anchored proteins such as CD55, CD59and CD16. (Yokochi (2001) Journal of Interferon and Cytokine Research21:389-398; Yamada (2004) The Journal of Immunology 172: 3869-3875).Propagermanium inhibits in-vitro chemotactic migration of monocytes byMCP-1 (Yokuchi (2001) Journal of Interferon and Cytokine Research21:389-398).

The invention provides a pharmaceutical composition comprising:

-   -   a) at least one angiotensin receptor blocker or a        pharmaceutically acceptable salt thereof; and    -   b) propagermanium or a pharmaceutically acceptable salt thereof.

The invention provides a pharmaceutical composition comprising:

-   -   a) at least one angiotensin receptor blocker or a        pharmaceutically acceptable salt thereof; and    -   b) RS504393, or a pharmaceutically acceptable salt thereof.

Key complement regulators CD55 (decay-accelerating factor) and CD59(protectin) are both GPI-anchored plasma membrane proteins (Yokochi(2001) Journal of Interferon and Cytokine Research 21:389-398; Yamada(2004) The Journal of Immunology 172: 3869-3875). Defective regulationof complement inhibitors and reduced levels of CD55 and CD59 have beenshown in a number of disease states including:

-   -   i) kidney diseases and renal ischemia reperfusion injury        (Yamada (2004) Critical Protection from Renal Ishemia        Reperfusion Injury by CD55 and CD59, The Journal of Immunology        172: 3869-3875);    -   ii) diabetes, where defective regulation of complement        inhibitors and reduced levels of CD55 and CD59 may be viewed as        a primary effect of diabetes and one of the mechanisms for        complement activation in diabetic vessels with the selective        decrease in these GPI-anchored complement inhibitors suggesting        effects of diabetes on common regulatory steps in the synthesis        or the processing of these molecules. It has been proposed that        the mechanism that leads to decreased levels of CD59 and CD55 in        diabetes may be cell- or tissue-specific. (Zhang (2002) Diabetes        51:3499-3504).    -   iii) macrovascular diseases (Ma (2009) Chinese medical journal,        122(18) 2123-2128);    -   iv) macular degeneration (Bora (2007) The Journal of Immunology,        178 (3) 1783-1790; and Ma K (2010) Invest Ophthalmol Vis Sci.        December; 51(12):6776-83. Epub 2010 August)

The phrase “angiotensin receptor” or “ATR” is to be understood to meaneither angiotensin receptor 1 (AT1R; AT₁R) or angiotensin receptor 2(AT2R; AT₂R), being G protein-coupled receptors. In one preferredembodiment, they are analogous to those described by Porrello et al.(Porrello, E. R. et al (2009) Frontiers in Bioscience, 14:958-972),which are activated by angiotensin II (AngII) and/or angiotensin III(AngIII). “Angiotensin receptor” or “ATR” is to be further understood toinclude newly discovered angiotensin receptor family members.

The phrase “angiotensin receptor blocker” is understood to mean an agentor compound which can inhibit or partially inhibits the activation ofthe ATR. This includes antagonists for ATR, inverse agonists andnegative allosteric modulators. Preferably, the angiotensin receptorblocker blocks AT1R.

The term “inhibits”, as used herein, means a reduction below detectablelimits when compared to a reference. The phrase includes blocking,retarding, or impeding an action to prevent an undesirable result.

The term “partially inhibits” as used herein, means any reduction withindetectable limits when compared to a reference. The phrase includesblocking, retarding, or impeding an action to prevent an undesirableresult.

The inhibition or partial inhibition may be measured using the in vitromethods set out herein, and include but are not limited to, biochemicalor cellular assays for the assessment of in vitro chemotactic migrationof monocytes by MCP-1 such as are known in the art, as well asmeasurement of inositol phosphate production, extracellular-regulatedkinase (ERK) phosphorylation, cAMP production, label-free technologies(such as using impedance, light refraction or charge redistribution), Gprotein coupling using proximity reporter systems or other approaches,β-arrestin recruitment or mediated signalling, transcriptionfactor-based reporter systems, microscopy visualization usingfluorescent labels, use of antibodies to assess receptor cellularlocalization (such as enzyme-linked immunosorbent assays) andfluorescence activated cell sorting.

The inhibition or partial inhibition may be measured using the in vivomethods set out herein, and include but are not limited to, serialmeasurements of renal function made by the measurement of plasmacreatinine and urea such as by way of an autoanalyser; the measurementof proteinuria, the measurement of albuminuria such as by way of aradioimmunoassay; and GFR (single shot isotopic technique); theassessment of endpoints such as renal and/or cardiac and/or ocularstructure, by way of, for example, light microscopy (LM) for theassessment of glomerular and cardiac hypertrophy, glomerulosclerosisand/or fibrosis and/or podocyte change and/or; immunohistochemistry tomeasure the extent of matrix deposition and modulation of profibroticgrowth factors and their activity; assessment of systolic bloodpressure, modulation of insulin fasting plasma glucose, modulation foHemoglobin A1c; and molecular biological techniques to assess renal andcardiac and ocular structure according to conventional assays such asknown in the art. Inhibition or partial inhibition may be indicated by aqualitative improvement in renal and/or cardiac and/or ocular structureas measured by one or more of the above mentioned endpoints.

The term “component” as used herein in the context of a pharmaceuticalcomposition of the invention, means either the angiotensin receptorblocker or the chemokine receptor pathway inhibitor.

In one preferred embodiment the angiotensin receptor blocker is selectedfrom the group consisting of:

-   -   a) an angiotensin receptor antagonist;    -   b) an angiotensin receptor inverse agonist; or    -   c) an angiotensin receptor negative allosteric modulator.

In a further preferred embodiment the angiotensin receptor blocker isselected from the group consisting of: CGP-42112A (AT₂R antagonist;Sigma # C-160), Eprosartan (AT₁R; market name Teveten®, AbbottLaboratories USA), Losartan (AT₁R; market name Cozaar®, Merck & Co),Valsartan (AT₁R; market name Diovan®, Novartis), Telmisartan (AT₁R,market name Micardis®, Boehringer Ingelheim), Irbesartan (AT₁R, marketname Avapro®, SanofiAventis), Candesartan (AT₁R, market name Atacand®,AstraZenica), Olmesartan (AT₁R, market name Benicar@, Daiichi SankyoInc), PD123319 (AT₂R, Tocris), ZD-7115 (AT1R), Saralasin((Sar¹-Ala⁸)AngII), Sarthran ((Sar¹-Thr⁸)AngII) and DuP753 (AT₁R). As anexample, the angiotensin receptor blocker may be irbesartan. Theangiotensin receptor blocker may be selected from the group comprisingCGP-42112A (AT₂R antagonist), Eprosartan (AT₁R), Losartan (AT₁R),Valsartan (AT1R), Telmisartan (AT₁R), Irbesartan (AT₁R), Candesartan(AT₁R), Olmesartan (AT₁R), PD123319 (AT₂R), ZD-7115 (AT₁R), Saralasin((Sar¹-Ala⁸)AngII), Sarthran ((Sar¹-Thr⁸)AngII) and DuP753 (AT₁R).

Irbesartan is an Angiotensin II receptor antagonist also known as2-butyl-3-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1,3-diazaspiro[4.4]non-1-en-4-one.Irbesartan has the following formula:

In one preferred embodiment the angiotensin receptor blocker is notOlmesartan. In another preferred embodiment the angiotensin receptorblocker is not Olmesartan and the chemokine receptor pathway inhibitoris not RS102895. In another preferred embodiment the angiotensinreceptor blocker is Olmesartan and the chemokine receptor pathwayinhibitor is Propagermanium. In another preferred embodiment theangiotensin receptor blocker is Olmesartan and the chemokine receptorpathway inhibitor is a chemokine receptor pathway inhibitor whichtargets a component of the chemokine receptor pathway other than thechemokine receptor.

Olmesartan is an Angiotensin II receptor antagonist also known as(5-methyl-2-oxo-2H-1,3-dioxol-4-yl)methyl4-(2-hydroxypropan-2-yl)-2-propyl-1-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl}methyl)-1H-imidazole-5-carboxylate.

Olmesartan has the following formula:

The invention therefore also provides a pharmaceutical compositioncomprising:

-   -   a) Olmesartan or a pharmaceutically acceptable salt thereof; and    -   b) at least one chemokine receptor pathway inhibitor or a        pharmaceutically acceptable salt thereof which inhibits a        component of the chemokine receptor pathway other than the        chemokine receptor.

The invention therefore also provides a pharmaceutical compositioncomprising:

-   -   a) at least one angiotensin receptor blocker or a        pharmaceutically acceptable salt thereof; and    -   b) propagermanium or a pharmaceutically acceptable salt thereof.

The invention therefore also provides a pharmaceutical compositioncomprising:

-   -   a) Olmesartan or a pharmaceutically acceptable salt thereof; and    -   b) propagermanium or a pharmaceutically acceptable salt thereof.

The invention therefore also provides a pharmaceutical compositioncomprising:

-   -   a) irbesartan or a pharmaceutically acceptable salt thereof; and    -   b) at least one chemokine receptor pathway inhibitor or a        pharmaceutically acceptable salt thereof.

The invention further provides a pharmaceutical composition comprising:

-   -   a) irbesartan or a pharmaceutically acceptable salt thereof; and    -   b) RS504393, or a pharmaceutically acceptable salt thereof.

The invention further provides a pharmaceutical composition comprising:

-   -   a) irbesartan or a pharmaceutically acceptable salt thereof; and    -   b) at least one chemokine receptor pathway inhibitor or a        pharmaceutically acceptable salt thereof which inhibits a        component of the chemokine receptor pathway other than the        chemokine receptor.

In one preferred embodiment, the total efficacy of the pharmaceuticalcomposition is greater when compared to the efficacies of theangiotensin receptor blocker or the chemokine receptor pathway inhibitorwhen either component is administered without any administration of theother component. Thus, the combined composition may be administered in asingle dose, including at sub-therapeutic doses, or less often, thaneither of the two components might be administered as single compounds.

Preferably, the total efficacy of the pharmaceutical composition isgreater when compared to the sum of the efficacies of the angiotensinreceptor blocker and the chemokine receptor pathway inhibitor wheneither component is administered without any administration of the othercomponent. More preferably, a synergistic effect in efficacy is observedwhen the angiotensin receptor blocker and the chemokine receptor pathwayinhibitor are administered concurrently or sequentially.

Alternatively, the total efficacy of the pharmaceutical composition isequal to the sum of the efficacies of the angiotensin receptor blockerand the chemokine receptor pathway inhibitor when either component isadministered without any administration of the other component. As afurther preferred embodiment of this alternative, an additive effect inefficacy is observed when the angiotensin receptor blocker and thechemokine receptor pathway inhibitor are administered concurrently orsequentially.

In a further alternative, the total efficacy of the pharmaceuticalcomposition is less than the sum of the efficacies of the angiotensinreceptor blocker and the chemokine receptor pathway inhibitor wheneither component is administered without any administration of the othercomponent. In a further embodiment, while the combined efficacy is lessthan the sum of the efficacies of the angiotensin receptor blocker andthe chemokine receptor pathway inhibitor when each component isadministered without any administration of the other component, thetreatment provides greater efficacy compared to a single treatment ofangiotensin receptor blocker or the chemokine receptor pathway inhibitoradministered alone.

Preferably the two components are administered concurrently at the sametime (for example as two tablets taken together, or as a single tablet,formulated with each component) or sequentially (for example one tablettaken after another tablet). The doses of each component may be takentogether (concurrently), or sequentially and taken within seconds,minutes, days, weeks or months of each other.

Method of Treatment

The present invention further provides a method for the treatment,amelioration or prevention of a condition or disease comprisingadministering to said subject a therapeutically effective amount ofcombination of (i) an angiotensin receptor blocker and (ii) an inhibitorof the chemokine receptor or its downstream pathways (a chemokinereceptor pathway inhibitor).

The inhibitor of the chemokine receptor or its downstream pathways (thechemokine receptor pathway inhibitor) and the angiotensin receptorblocker used in the method of the present invention may be chosen fromthose discussed above.

Preferably, the condition or disease to be treated or prevented is akidney disease, more particularly a disease selected from the groupconsisting of: fibrotic disorders in the kidney, chronic kidney diseasecaused by diabetic nephropathy, renal insufficiency (diabetic andnon-diabetic), and renal failure conditions, including diabeticnephropathy, glomerulonephritis, scleroderma, glomerular sclerosis,proteinuria of primary renal disease and renal vascular hypertension.

The method of the invention may also be used to treat or prevent acondition selected from the group consisting of: cardiovascular diseaseincluding, (acute and chronic) congestive heart failure, leftventricular dysfunction and hypertrophic cardiomyopathy, diabeticcardiac myopathy, supraventricular and ventricular arrhythmias, atrialfibrillation or atrial flutter, myocardial infarction and its sequelae,atherosclerosis, angina (whether unstable or stable), heart failure,angina pectoris, diabetes, secondary aldosteronism, primary andsecondary pulmonary hyperaldosteronism, primary and pulmonaryhypertension, diabetic retinopathy, macular degeneration, oculardisorders, insulin resistance, the management of other vasculardisorders, such as migraine, Raynaud's disease, luminal hyperplasia,cognitive dysfunction (such as Alzheimer's), stroke, hyperkalemia,preeclampsia, sarcoidosis, HIV infection and AIDS pathogenesis, ischemiaand reperfusion injury, atherogenesis, chronic obstructive pulmonarydisease, asthma and allergy renal disease, rheumatoid arthritis.

Generally, a range of ailments which are chemokine-related may betreated by the method of the present invention, including ailments thatare related to increased or decreased production of chemokines, and/orincreased or decreased responsiveness of cells to chemokines. Achemokine-related ailment should also be understood to mean a conditionin which chemokine receptors display aberrant characteristics, are thetarget of a particular pathogen or are a target of a pharmacologicalintervention. The following list provides some examples ofchemokine-related ailments:

-   -   HIV infection and AIDS pathogenesis    -   Ischemia and reperfusion injury;    -   Atherogenesis;    -   Chronic obstructive pulmonary disease;    -   Asthma and allergy;    -   Renal disease    -   Rheumatoid arthritis

However, it should be understood that the phrase ‘chemokine-relatedinterventions’ and the phrase ‘a chemokine-related ailment’ is notlimited thereto.

A range of ailments which are related to angiotensin may also be treatedby the method of the present invention, including ailments that arerelated to increased or decreased production of angiotensin, and/orincreased or decreased responsiveness of cells to angiotensin. Listedbelow are a number of conditions that have either been proposed to stemfrom a dysregulated angiotensin system, or, could potentially be treatedusing angiotensin-based interventions:

-   -   Chronic heart failure;    -   Atherosclerosis/ischemia;    -   Hypertension;    -   Hyperkalemia;    -   Preeclampsia;    -   Diabetes mellitus;    -   Diabetic retinopathy;    -   Sarcoidosis;    -   Alzheimer's Disease

The condition or disease to be treated or prevented may be a diseaseselected from the group comprising fibrotic disorders in the kidney,chronic kidney disease caused by diabetic nephropathy, renalinsufficiency, renal failure conditions, diabetic nephropathy,glomerulonephritis, scleroderma, glomerular sclerosis, proteinuria ofprimary renal disease, renal vascular hypertension, cardiovasculardisease, chronic heart failure, hypertension, congestive heart failure,left ventricular dysfunction, hypertrophic cardiomyopathy, diabeticcardiac myopathy, supraventricular and ventricular arrhythmias, atrialfibrillation, atrial flutter, myocardial infarction and its sequelae,atherosclerosis, angina, heart failure, angina pectoris, secondaryaldosteronism, primary and secondary pulmonary hyperaldosteronism,primary and pulmonary hypertension, diabetic retinopathy, maculardegeneration, ocular disorders, insulin resistance, the vasculardisorders, migraine, Raynaud's disease, luminal hyperplasia, cognitivedysfunction, Alzheimer's disease, stroke, hyperkalemia, preeclampsia,sarcoidosis, Diabetes mellitus; Diabetic retinopathy, HIV infection,AIDS pathogenesis, ischemia and reperfusion injury, atherogenesis,chronic obstructive pulmonary disease, asthma, allergy renal disease,and rheumatoid arthritis.

In one aspect, the chemokine receptor pathway inhibitor inhibits orpartially inhibits a protein other than the chemokine receptor, morepreferably, the inhibitor is an agent which blocks MCP-1 inducedmigration and activation of monocytes and chemotactic migration throughthe targeting of glycosylphosphatidylinositol (GPI)-anchored proteinssuch as CD55, CD59 and CD16. Most preferably, the chemokine receptorpathway inhibitor is propagermanium.

While not intending to be restricted to any particular mode of action,in one preferred embodiment the chemokine receptor inhibitor has agreater affinity and/or potency and/or efficacy when interacting withthe chemokine receptor or modulating its downstream pathways when thechemokine receptor is associated with the angiotensin receptor. Forexample, the chemokine receptor and the angiotensin receptor may beassociated as a chemokine receptor/angiotensin receptorhetero-dimer/-oligomer. In a further preferred embodiment, when thechemokine receptor inhibitor is administered to a subject concurrentlyor sequentially with an angiotensin receptor blocker, the combinedaffinity, potency and/or efficacy is greater than compared to theaffinity, potency and/or efficacy that would have been achieved when thechemokine receptor inhibitor is not administered in combination (whetherconcurrently or sequentially) with the angiotensin receptor blocker. Inan even further preferred embodiment, a synergistic effect (as measuredby affinity, potency and/or efficacy) is achieved when the chemokinereceptor inhibitor is administered to a subject in combination (whetherconcurrently or sequentially) with an angiotensin receptor blocker.

While not intending to be restricted to any particular mode of action,in one preferred embodiment the angiotensin receptor blocker has agreater affinity and/or potency and/or efficacy when interacting withthe angiotensin receptor when the angiotensin receptor is associatedwith the chemokine receptor. For example, the chemokine receptor and theangiotensin receptor may be associated as a chemokinereceptor/angiotensin receptor hetero-dimer/-oligomer. In a furtherpreferred embodiment, when the angiotensin receptor blocker isadministered to a subject concurrently or sequentially with a chemokinereceptor inhibitor, the combined affinity, potency and/or efficacy isgreater than compared to the affinity, potency and/or efficacy thatwould have been achieved when the angiotensin receptor blocker is notadministered in combination (whether concurrently or sequentially) withthe chemokine receptor inhibitor. In an even further preferredembodiment, a synergistic effect (as measured by affinity, potencyand/or efficacy) is achieved when the angiotensin receptor blocker isadministered to a subject in combination (whether concurrently orsequentially) with a chemokine receptor inhibitor.

Manufacture of a Medicament

The invention also provides for the use of a pharmaceutical compositioncomprising at least one angiotensin receptor blocker or apharmaceutically acceptable salt thereof, and at least one chemokinereceptor pathway inhibitor or a pharmaceutically acceptable saltthereof; for the manufacture of a dosage form for the treatment of adisease. The pharmaceutical composition may optionally further comprisea pharmaceutically acceptable carrier.

Dosage Forms, Formulations and Administration

The dosage form provided by the present invention may further comprise avial, cartridge, container, tablet or capsule comprising thepharmaceutical composition of the invention together with dosageinstructions for the administration of the dosage form to a subject forthe treatment, amelioration or prevention of a disease.

Dosage levels of the compounds of the invention will usually be of theorder of about 0.5 mg to about 20 mg per kilogram body weight, with apreferred dosage range between about 0.5 mg to about 10 mg per kilogrambody weight per day (from about 0.5 g to about 3 g per patient per day).The amount of each active ingredient which may be combined with thecarrier materials to produce a single dosage will vary, depending uponthe host to be treated and the particular mode of administration. Forexample, a formulation intended for oral administration to humans maycontain about 5 mg to 1 g of each active compound with an appropriateand convenient amount of carrier material, which may vary from about to95 percent of the total composition. Dosage unit forms will generallycontain between from about 5 mg to 500 mg of active ingredient.

Preferably, the angiotensin receptor blocker is provided at a dose ofbetween 50 mg to 500 mg per day. Even more preferably, the chemokinereceptor pathway inhibitor is provided at a dose of between 150 mg to300 mg per day. For example, the angiotensin receptor blocker isIrbesartan and is administered at a dose of 300 mg per day.

Preferably, the chemokine receptor pathway inhibitor is provided at adose of between 5 mg to 2000 mg per day. Even more preferably thechemokine receptor pathway inhibitor is provided at a dose of between 5mg to 50 mg per day. For example, the chemokine receptor pathwayinhibitor is propagermanium and is provided at a dose of 30 mg per day.

The dosage form may comprise about 5 mg to 1 g of the angiotensinreceptor blocker or a pharmaceutically acceptable salt thereof, andabout 5 mg to 1 g of the chemokine receptor pathway inhibitor or apharmaceutically acceptable salt thereof. The dosage form may comprise adaily dose of angiotensin receptor blocker of between about 50 mg to 500mg. The angiotensin receptor blocker may be Irbesartan, and the dosageform may comprise a daily dose of Irbesartan of about 300 mg. The dosageform may also comprise a daily dose of chemokine receptor pathwayinhibitor of between about 5 mg to 50 mg. The chemokine receptor pathwayinhibitor may be propagermanium and the dosage form may comprise a dailydose of propagermanium of about 30 mg

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

Medicaments of the invention, in various aspects, may be administered byinjection, or prepared for oral, pulmonary, nasal or for any other formof administration. Preferably the medicaments are administered, forexample, intravenously, subcutaneously, intramuscularly, intraorbitally,ophthalmically, intraventricularly, intracranially, intracapsularly,intraspinally, intracisternally, intraperitoneally, buccal, rectally,vaginally, intranasally or by aerosol administration.

The mode of administration is in one aspect at least suitable for theform in which the medicament has been prepared. The mode ofadministration for the most effective response is in one aspectdetermined empirically and the means of administration described beloware given as examples, and do not limit the method of delivery of thecomposition of the present invention in any way. All the aboveformulations are commonly used in the pharmaceutical industry and arecommonly known to suitably qualified practitioners.

The medicaments of the invention in certain aspects may includepharmaceutically acceptable nontoxic excipients and carriers andadministered by any parenteral techniques such as subcutaneous,intravenous and intraperitoneal injections. In addition the formulationsmay optionally contain one or more adjuvants. As used herein, a“pharmaceutical carrier” is a pharmaceutically acceptable solvent,suspending agent, excipient or vehicle for delivering the compounds tothe subject. The carrier may be liquid or solid, and is selected withthe planned manner of administration in mind.

The pharmaceutical forms suitable for injectable use optionally includesterile aqueous solutions (where water-soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. Alternatively, the compounds of the inventionare, in certain aspects encapsulated in liposomes and delivered ininjectable solutions to assist their transport across cell membrane.Alternatively or in addition such preparations contain constituents ofself-assembling pore structures to facilitate transport across thecellular membrane. The carrier, in various aspects, is a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. Properfluidity is maintained, for example and without limitation, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of the injectable compositions is in certainaspects brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

The invention also provides an injectable sustained releasepharmaceutical composition comprising a therapeutically effectivepharmaceutical composition according to the invention, and a releaseretardant. The release retardant may be, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in an appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the various sterilised active ingredient into a sterilevehicle that contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, preparation incertain aspects include without limitation vacuum drying andfreeze-drying techniques that yield a powder of the active ingredientplus any additional desired ingredient from previously sterile-filteredsolution thereof.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Martin, Remington's Pharmaceutical Sciences, 18thEd. (1990 Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatised with various polymers (E.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, in Modern Pharmaceutics, Chapter 10,Banker and Rhodes ed., (1979), herein incorporated by reference. Ingeneral, the formulation will include the compounds described as part ofthe invention (or a chemically modified form thereof), and inertingredients which allow for protection against the stomach environment,and release of the biologically active material in the intestine.

For the chemokine receptor pathway inhibitor or angiotensin receptorblocker of the invention the location of release may be the stomach, thesmall intestine (the duodenum, the jejunum, or the ileum), or the largeintestine. One skilled in the art has available formulations that willnot dissolve in the stomach, yet will release the material in theduodenum or elsewhere in the intestine. In one aspect, the release willavoid the deleterious effects of the stomach environment, either byprotection of the composition or by release of the compounds beyond thestomach environment, such as in the intestine.

The invention further provides an oral sustained release pharmaceuticalcomposition comprising a therapeutically effective pharmaceuticalcomposition according to the invention, and a release retardant.

In one aspect of the present invention the release retardant is awater-soluble, water swellable and/or water insoluble polymer. Inparticular, water-soluble polymers are selected from the groupcomprising are ethylcellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, an enteric coating; and a semipermeable membrane. Inanother aspect of the invention the release retardant is a non-polymericrelease retardant. More particularly, the non-polymeric releaseretardant is hydrogenated castor oil. The compositions of the inventionmay be milled or granulated and compressed into tablets or encapsulatedinto capsules according to conventional procedures known in the art.

To ensure full gastric resistance, a coating impermeable to at least pH5.0 is used. Examples of the more common inert ingredients that are usedas enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This includes withoutlimitation sugar coatings, or coatings that make the tablet easier toswallow. Exemplary capsules consist of a hard shell (such as gelatin)for delivery of dry therapeutic i.e. powder; for liquid forms, a softgelatine shell may be used. The shell material of cachets in certainaspects is thick starch or other edible paper. For pills, lozenges,moulded tablets or tablet triturates, moist massing techniques are alsocontemplated, without limitation.

As used herein, the term “sustained release” means the gradual butcontinuous or sustained release over a relatively extended period of thetherapeutic compound content after oral ingestion. The release maycontinue after the pharmaceutical composition has passed from thestomach and through until and after the pharmaceutical compositionreaches the intestine. The phrase “sustained release” also means delayedrelease wherein release of the therapeutic compound is not immediatelyinitiated upon the pharmaceutical composition reaching the stomach butrather is delayed for a period of time, for example, until when thepharmaceutical composition reaches the intestine. Upon reaching theintestine, the increase in pH may then trigger release of thetherapeutic compound from the pharmaceutical composition.

Though term “release retardant” is used herein, means a substance thatreduces the rate of release of a therapeutic compound from apharmaceutical composition when orally ingested. The release retardantmay be a polymer or a non-polymer. The release retardant may be usedaccording to any one of several sustained release systems including, forexample, a diffusion system, a dissolution system and/or an osmoticsystem.

In certain aspects, the therapeutic is included in the formulation asfine multiparticulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the material for capsuleadministration is, in certain aspects, a powder, lightly compressedplugs or even as tablets. In one aspect, the therapeutic could beprepared by compression.

Colourants and flavouring agents are optionally all be included. Forexample, compounds may be formulated (such as, and without limitation,by liposome or microsphere encapsulation) and then further containedwithin an edible product, such as a refrigerated beverage containingcolorants and flavouring agents.

The volume of the therapeutics, in one aspect, diluted or increased withan inert material. These diluents could include carbohydrates,especially mannitol, alpha-lactose, anhydrous lactose, cellulose,sucrose, modified dextrans and starch. Certain inorganic salts are alsooptionally used as fillers including calcium triphosphate, magnesiumcarbonate and sodium chloride. Some commercially available diluents areFast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

In other embodiments, disintegrants are included in the formulation ofthe therapeutic into a solid dosage form. Materials used asdisintegrants include but are not limited to starch including thecommercial disintegrant based on starch, Explotab. Sodium starchglycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,sodium alginate, gelatine, orange peel, acid carboxymethyl cellulose,natural sponge and bentonite are also contemplated. Another form of thedisintegrants is the insoluble cationic exchange resins. Powdered gumsare also optionally used as disintegrants and as binders and theseinclude, without limitation, powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders are contemplated to hold the therapeutic compounds together toform a hard tablet and include, without limitation, materials fromnatural products such as acacia, tragacanth, starch and gelatin. Otherbinders include, without limitation, methylcellulose (MC), ethylcellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone(PVP) and hydroxypropylmethyl cellulose (HPMC) are contemplated for usein alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be optionally included in the formulation ofthe therapeutic to prevent sticking during the formulation process.Lubricants may be optionally used as a layer between the therapeutic andthe die wall, and these can include but are not limited to: stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Exemplary solublelubricants may also be used such as include sodium lauryl sulfate,magnesium lauryl sulfate, polyethylene glycol of various molecularweights, and Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the compound duringformulation and to aid rearrangement during compression might beoptionally added. The glidants may include without limitation starch,talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment, asurfactant might be added in certain embodiments as a wetting agent.Surfactants may include, for example and without limitation, anionicdetergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinateand dioctyl sodium sulfonate. Cationic detergents might be optionallyused and could include, without limitation, benzalkonium chloride orbenzethomium chloride. The list of potential nonionic detergents thatcould be included in the formulation as surfactants are lauromacrogol400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10,50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrosefatty acid ester, methyl cellulose and carboxymethyl cellulose. Whenused, these surfactants could be present in the formulation of thecompounds either alone or as a mixture in different ratios.

Additives which that potentially enhance uptake of the compounds are forinstance and without limitation the fatty acids oleic acid, linoleicacid and linolenic acid.

Controlled release formulation may be desirable. The formulations arealso contemplated. In certain aspects, the compounds could beincorporated into an inert matrix that permits release by eitherdiffusion or leaching mechanisms i.e., gums. In some aspects, slowlydegenerating matrices may also be incorporated into the formulation.Another form of a controlled release of this therapeutic is by a methodbased on the Oros therapeutic system (Alza Corp.), i.e. the drug isenclosed in a semipermeable membrane which allows water to enter andpush drug out through a single small opening due to osmotic effects.Some enteric coatings also have a delayed release effect.

In other aspects, a mix of materials might be used to provide theoptimum film coating. Film coating may be carried out, for example andwithout limitation, in a pan coater or in a fluidized bed or bycompression coating.

Also contemplated herein is pulmonary delivery of the compounds. Inthese aspects, the compounds may be delivered to the lungs of a mammalwhile inhaling and traverses across the lung epithelial lining to theblood stream.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered-doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Some specific examples of commercially available devices suitable forthe practice of this invention are, for example and without limitation,the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis,Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products,Englewood, Colo.; the Ventolin metered dose inhaler, manufactured byGlaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powderinhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the compounds. Typically, each formulation is specific tothe type of device employed and may involve the use of an appropriatepropellant material, in addition to the usual diluents, adjuvants and/orcarriers useful in therapy. Also, the use of liposomes, microcapsules ormicrospheres, inclusion complexes, or other types of carriers iscontemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the compounds suspended in water.The formulation may also include, in one aspect, a buffer and a simplesugar (e.g., for protein stabilization and regulation of osmoticpressure). In one embodiment, the nebulizer formulation may also containa surfactant, to reduce or prevent surface induced aggregation of thecompounds caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise, in one aspect a finely divided powder containing the compoundssuspended in a propellant with the aid of a surfactant. The propellantmay be is any conventional material employed for this purpose, such asand without limitation, a chlorofluorocarbon, a hydrochlorofluorocarbon,a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude, without limitation sorbitan trioleate and soya lecithin. Oleicacid may also be useful as a surfactant in certain aspects.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the compound and may also include abulking agent, such as and without limitation lactose, sorbitol,sucrose, or mannitol in amounts which facilitate dispersal of the powderfrom the device, e.g., 50 to 90% by weight of the formulation. Incertain embodiments, the compound(s) is/are prepared in particulate formwith an average particle size of less than 10 microns, most preferably0.5 to 5 microns, for most effective delivery to the distal lung.

Nasal delivery of the compounds is also contemplated. Nasal deliveryallows the passage of the protein to the blood stream directly afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with, for example and without limitation, dextranor cyclodextran.

It will be appreciated that in certain aspects, the medicaments of theinvention may be given as a single dose schedule, or preferably, in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of delivery may be with 1 to 10 separate doses, isoptionally followed by other doses given at subsequent time intervalsrequired to maintain or reinforce the treatment. The dosage regimen willalso, at least in part, be determined by the needs of the individual andthe judgement of the practitioner.

The invention thus provides a tablet comprising the pharmaceuticalcomposition of the invention; a capsule comprising the pharmaceuticalcomposition of the invention and injectable suspension comprising thepharmaceutical composition of the invention, and a composition forpulmonary delivery comprising the pharmaceutical competition of theinvention.

Assessing the Efficacy of the Pharmaceutical Compositions

In another aspect of the invention, there is provided a method forassessing the efficacy of a pharmaceutical composition of the invention,wherein the method includes a step selected from the group including:assessing the in vitro chemotactic migration of monocytes by MCP-1 byway of an in vitro biochemical or cellular assay; measuring inositolphosphate production, extracellular-regulated kinase (ERK)phosphorylation or cAMP production; measuring the effect of thecomposition using label-free technologies, such as using impedance,light refraction or charge redistribution; measuring G protein couplingusing proximity reporter systems or other approaches; measuringβ-arrestin recruitment or mediated signalling; measuring the effect ofthe composition using transcription factor-based reporter systems;utilizing in vitro biochemical or cellular techniques to measurecellular localization, such as microscopy visualization usingfluorescent labels, use of antibodies (such as enzyme-linkedimmunosorbent assays) and fluorescence activated cell sorting; measuringthe in vivo levels of plasma creatinine and urea, as indicative of renalfunction, such as by way of an autoanalyser; measuring the levels ofproteinuria, measuring the levels of albuminuria by way of aradioimmunoassay; measuring GFR (single shot isotopic technique);assessing renal and/or cardiac and/or ocular or other tissue structureby way of light microscopy (LM); assessing the presence and/or extent ofglomerular and/or cardiac hypertrophy, glomerulosclerosis and/orfibrosis; assessing the extent of matrix deposition, assessing themodulation of profibrotic growth factors and their activity; assessingrenal and/or cardiac structure and/or ocular structure and/or othertissue structure; and assessing systolic blood pressure, modulation ofinsulin fasting plasma glucose, and/or modulation of Hemoglobin A1c.

In a further aspect of the invention, there is provided a method forassessing the inhibition or partial inhibition activity of apharmaceutical composition of the invention, wherein the inhibition orpartial inhibition is indicated by a qualitative improvement in renaland/or cardiac and/or ocular and/or other tissue structure as measuredby one or more of the following: levels of plasma creatinine and urea;levels of proteinuria, levels of albuminuria; GFR (using single shotisotopic technique); integrity of renal and/or cardiac and/or ocularstructure; the extent of matrix deposition; modulation of the activityof profibrotic growth factors; light microscopy (LM) for the assessmentof glomerular and/or cardiac hypertrophy, glomerulosclerosis and/orfibrosis; immunohistochemistry to measure the extent of matrixdeposition and modulation of profibrotic growth factors and theiractivity; and molecular biological techniques to assess renal and/orcardiac and/or ocular structure.

The invention will now be further described by way of reference only tothe following non-limiting examples. It should be understood, however,that the examples following are illustrative only, and should not betaken in any way as a restriction on the generality of the inventiondescribed above.

EXAMPLES Example 1—Reduction of Proteinuria in STNx Model (Low DosePropagermanium)

Sub-Total Nephrectomy (STNx) Surgery

Animal

Six weeks old, male Sprague-Dawley (SD) rats weighing 200-250 g aresourced from Animal Resources Centre (Western Australia). The animalstudy is conducted with the approval from the Animal Ethics Committee(St Vincent's Hospital and the National Health and Medical ResearchFoundation of Australia). All rats receive normal rat chow (CertifiedRodent Diet #5002, LabDiet, USA) and drinking water ad libitum. Allanimals are housed in a stable environment maintained at 22±1° C. with a12-hour light/dark cycle commencing at 6 am. STNx surgery is performedin operating theater at St Vincent's Experimental Surgical Unit. Allsurgical procedures are modified from those previously described(Gilbert, R. E., L. L. Wu, et al. (1999). “Pathological expression ofrenin and angiotensin II in the renal tubule after subtotal nephrectomy.Implications for the pathogenesis of tubulointerstitial fibrosis.” Am JPathol 155(2): 429-40.; Kelly, D. J., A. J. Edgley, et al. (2009).“Protein kinase C-beta inhibition attenuates the progression ofnephropathy in non-diabetic kidney disease.” Nephrol Dial Transplant24(6): 1782-90.; Kelly, D. J., C. Hepper, et al. (2003). “Vascularendothelial growth factor expression and glomerular endothelial cellloss in the remnant kidney model.” Nephrol Dial Transplant 18(7):1286-92.; Wu, L., A. Cox, et al. (1997). “Transforming growth factor 31and renal injury following subtotal nephrectomy in the rat: Role of therenin-angiotensin system.” Kidney Int 51: 1553-1567.)

Pre-Operative Care

The afternoon before surgery, the rats are weighed and given one dose ofantibiotics (oxytetracycline, 30 mg/kg) as prophylaxis by oral gavage.The rats are then fasted overnight.

Operative Care

Anaesthesia

Anaesthesia is induced with 2.5% isoflurane mixed with oxygen in aPerspex plastic box.

Once anaesthetised, the rat is then laid on its back on a heat pad(maintained at 37° C.), and a facemask is then placed over the rat'snose and mouth to deliver isoflurane, maintaining anaesthesia with 1-2%isoflurane/97% oxygen in a tidal volume of 1 ml/100 g body weight.

Skin Preparation

The abdominal area of the rat is shaved from the sternum to the pelvicarea using clippers. The shaved area is cleaned 3 times withChlorhexidine in Alcohol 70%, in a circular motion, starting at theincision point (midline) and cleaning outwards.

Surgery

A fenestrated drape is placed over the incision site and a skin incisionis made from 10 mm below the sternum to 10 mm above the genitals with aNo. 23 scalpel blade. The muscle layer is then exposed and raised withtissue forceps to allow for an incision along the linea alba (the fasciajoining the muscle layers along the midline) to be made. This raising ofthe muscle layer prevents the intestines being accidentally damaged bysharp instruments.

Once a small hole is made in the muscle layer using a scalpel blade,fine scissors are used to complete the incision. With both incisionscomplete, gauze is placed over the drape surrounding the incision siteand 0.9% saline is used to moisten the gauze. Using moistened cottonbuds, the left kidney is located and raised onto the gauze. Under themicroscope, toothed forceps and cotton buds are then used to dissect fataway from the renal pelvis exposing the branches of renal arteries justbefore into the kidney. Individual branches of renal arteries (3-4) arethen isolated by blunt dissection using fine forceps. 4.0 silk is thenpassed under the arteries until enough arteries are isolated toincapacitate blood flow to ⅔ of the kidney, rendering this area dead.Once it is ascertained that there is no bleeding and ⅓ of the kidney isstill functioning, the kidney is placed back into the abdomen.

The right kidney is then exposed and the renal capsule removed. 4.0 silkis used to tie off the kidney at the renal pelvis, ligating the wholekidney and the ureter. Three knots are tied on one side, the silk weavedto the other side and a further three knots tied on the other side. Thekidney is then cut out. When it is ascertained that there is nobleeding, the vascular stump is placed back into the abdomen. The leftkidney can then be re-checked to make sure the colour change issufficient and the remaining ⅓ of the kidney is still functional. Bothleft and right ureters are checked without any damages, 2.0 mls of 0.9%saline is then placed into the abdomen to aid in re-hydrating the rat incase there was fluid loss while the cavity was open.

Wound Closure 5.0 dissolvable sutures (PGA—Polyglycolic Acid sutures)are then used to stitch the muscle layer in a continuous stitch. Theskin is then stitched with a continuous stitch of 4.0 silk. The area isthen cleaned with Chlorhexidine in Alcohol 70% to remove any blood fromthe skin. The anaesthetic mask is then removed and Op-site spray (tissuespray) is sprayed onto the incision site to add an extra barrier forprotection against infection. While the rat is beginning to wake fromanaesthesia, Buprenorphine (Temgesic) is given at a dose of 0.03 mg/kgsubcutaneously.

Recovery Period

The rat is then allowed to recover on a heat pad maintained at 37° C.

Post-Operative Care

Post-operatively, a solution of 5% glucose is given in a drinking bottlealongside a water bottle in order to give the rats the option ofdrinking one or the other. Food is also placed in the bottom of the boxto facilitate eating. 12 to 24 hours post-surgery, if the rat is noteating or drinking, buprenorphine is administered at subcutaneously. 24to 48 hours post-surgery if the rat appears depressed, reluctant tomove, or is in a hunched position, this may be the result of renalfailure. In this case, the rats are culled using an overdose ofpentobarbitone sodium (120 mg/kg ip).

Every 4 weeks, systolic blood pressure (SBP) will be determined inpreheated conscious rats via tail-cuff plethysmography using anon-invasive blood pressure (NIBP) controller and Powerlab (ADinstruments, NSW, Australia).

Sham Surgery

The control rats will undergo sham surgery consisting of laparotomy asdescribed above and manipulation of both kidneys without dissectingrenal arteries before wound closure.

Study Design and Procedures

Treatments start 14 days following surgery.

Long term (12 weeks)

Untreated Groups

Groups 1, 2 Untreated: Control, Diabetic

Single Agent Group

Group 3 STNx+Irbesartan (ARB) (10 mg/kg/day)

Combination Group

Group 4 STNx+Irbesartan (10 mg/kg/day)/Propagermanium (3 mg/kg/day) n=16rats per group

Clinical Parameters

Serial measurements of systolic blood pressure (SBP) and clinicalparameters were undertaken at intervals as per standard protocols foranimal studies (every 4 weeks) (Kelly D J, Wilkinson-Berka J L, T. J. A,et al.: A new model of progressive diabetic renal impairment in thetransgenic (mRen-2)27 rat. Kidney Int. 54:343-352, 1998).

Renal and Cardiac Function (Primary Endpoints)

Serial measurements of renal function were made by the measurement ofplasma creatinine and urea (autoanalyser), albuminuria(radioimmunoassay, every 4 weeks) and GFR (single shot isotopictechnique, 4 and 12 weeks) as per standard protocols for animal studies(Kelly et al, 1998).

Future Experiments—Renal and Cardiac Structure (Secondary Endpoints)

Further experiments that could be performed include assessing secondaryendpoints such as renal and cardiac structure. For example, lightmicroscopy (LM) could be used to measure glomerular and cardiachypertrophy, glomerulosclerosis and fibrosis. Immunohistochemistry couldbe used to measure the extent of matrix deposition and modulation ofprofibrotic growth factors and their activity. Molecular biology couldalso be used to assess renal and cardiac structure.

Statistical Considerations

Comparisons between animal groups were performed using an ANOVA with aFishers post hoc test. The justification of animal usage has beencalculated to be n=16 rats per group (n=8 for Histology, n=8 formolecular biology). Values of p<0.05 were considered statisticallysignificant. Albuminuria was analysed following log transformation ofdata and geometric means x/÷tolerance factors.

As shown in FIG. 1, improved levels of proteinurea were achieved in thesub-total nephrectomy (STNx) model of end organ renal disease whenanimals were treated with a combination of an angiotensin receptorblocker and a low dose of propagermanium (STNx+Irb+PPG) as compared tountreated STNx animals and animals treated with the angiotensin receptorblocker alone (STNx+Irb).

Example 2—Reduction of Proteinuria in STNx Model (High DosePropagermanium)

As described for Example 1 but with the following treatments:

Study Design and Procedures

Treatments start 14 days following surgery.

Long term (12 weeks)

Untreated Groups

Groups 1, 2 Untreated: Control, Diabetic

Single Agent Group

Group 3 STNx+Irbesartan (ARB) (10 mg/kg/day) or STNx+Propagermanium (30mg/kg/day)

Combination Group

Group 4 STNx+Irbesartan (10 mg/kg/day)/Propagermanium (30 mg/kg/day)

n=16 rats per group

As shown in FIG. 2, improved levels of proteinurea were achieved in thesub-total nephrectomy (STNx) model of end organ renal disease whenanimals were treated with a combination of an angiotensin receptorblocker and a high dose of propagermanium (STNx+PPG+Irb) as compared tountreated STNx animals, STNx animals treated with a high dose ofpropagermanium alone (STNx+PPG), and STNx animals treated with theangiotensin receptor blocker alone (STNx+Irb).

Histological sections were obtained from the sub-total nephrectomy(STNx) model of end organ renal disease described above and stainedaccording to standard procedures to detect podocytes in the glomerulus.The histological sections were assessed by phase-contrast microscopy.FIG. 3 shows intense brown stained cells (podocytes) in the glomerulusof renal samples obtained from untreated control animals (A); STNxuntreated (B); STNx treated with propagermanium (C); STNx treated withIrbesartan (D); and STNx treated with propagermanium and Irbesartan incombination (E). FIG. 4 shows a bar graph of the improvement in numbersof podocytes detected. It can be seen that the level of podocytes inanimals treated with a combination of Irbesartan and propagermanium wasgreater than that of untreated animals subjected to the sub-totalnephrectomy (STNx) model of end organ renal disease and STNx animalstreated with either propagermanium or Irbesartan alone.

Example 3—Upon Co-Expression of AT1R and CCR2 Via Transient Transfectionof HEK293FT Cells, Combined Inhibition of Both Receptors Blocks ArrestinRecruitment to a Greater Extent than Inhibition of Either Receptor Alone

The combined effect of CCR2 and AT1R inhibition in vitro wasinvestigated by using RS504393 in combination with Irbesartan.

FIG. 5 shows the effect of AT1R and CCR2 blockade on β-arrestin2recruitment. HEK293FT cells were transiently transfected with plasmidscoding for β-arrestin2-Venus and the indicated receptors: CCR2-Rluc8(top panel), AT1R-Rluc8 (middle panel) or AT1R-Rluc8 and the untaggedCCR2 (bottom panel).

Cells were harvested 24 h post-transfection in HEPES-buffered phenolred-free complete medium containing 5% FCS and added to apoly-L-lysine-coated white 96-well plate. 48 h post-transfection, theplate was incubated at 37° C., 5% CO₂ for 2 hours with 30 μM EnduRen(Promega) to ensure substrate equilibrium was reached.

Cells were first pre-incubated or not for 30 minutes at 37° C. withIrbesartan (10 μM), RS504393 (10 μM) or both combined. Then cells werestimulated or not for 30 minutes at 37° C. with 100 nM of AngII, MCP-1or both together and the BRET signal was measured.

BRET detection was carried out in live cells by measuring sequentiallight emissions at 400-475 nm and 520-540 nm before and after agonistaddition. The BRET signal was calculated by subtracting the ratio of520-540 nm emission over 400-475 nm emission for a vehicle-treated cellsample from the same ratio for a second aliquot of the same cellstreated with ligand (ligand-induced BRET). Data represent mean±SEM ofthree independent experiments performed in triplicate.

As shown in FIG. 5 (top panel), 10 μM of RS504393 but not Irbesartansubstantially reduced the MCP-1-induced BRET signal between CCR2-Rluc8and 3-arrestin2-Venus. The combination of both antagonists did notsubstantially alter the inhibitory effect of RS504393 (FIG. 5: toppanel). Conversely, in cells co-expressing AT1R-Rluc8 and3-arrestin2-Venus, 10 μM of Irbesartan but not RS504393 substantiallyblocked the AngII-induced BRET response and their combination did notgive any different effect as expected (FIG. 5: middle panel).

However, in cells co-expressing AT1R-Rluc8, β-arrestin2-Venus anduntagged CCR2 where both AngII and MCP-1 induced BRET increases todifferent degrees, Irbesartan seems to substantially block the AngII-but not the MCP-1-induced BRET (FIG. 5: bottom panel). Similarly,RS504393 partially blocked the MCP-1- but not the AngII-promoted BRETsignal (FIG. 5: bottom panel). Importantly, the combination of bothantagonists reduced the BRET response to levels below that observed witheither individual antagonist alone, providing in vitro evidence for agreater inhibition of receptor-mediated cellular response, in this caseβ-arrestin recruitment, as a consequence of combined receptorinhibition.

Example 4—Upon Co-Expression of AT1R and CCR2 Via Transient Transfectionof HEK293FT Cells, Combined Inhibition of Both Receptors Blocks InositolPhosphate Signalling to a Greater Extent than Inhibition of EitherReceptor Alone

RS504393 was used in combination with Irbesartan to investigate thecombined effect of CCR2 and AT1R inhibition in vitro.

FIG. 6 shows the effect of AT1R and CCR2 blockade on inositol phosphateproduction. HEK293FT cells were transiently transfected with theplasmids coding for AT1R-Rluc8 and 3-arrestin2-Venus in the absence (toppanel) and presence (bottom panel) of untagged CCR2. 48 hpost-transfection, cells were used to generate the agonist-inducedinositol (1) phosphate (IP1) production measurements using the IP-One Tbkit (Cisbio Bioassays, Bagnol sur Ceze, France).

Cells were first pre-incubated or not for 30 minutes at 37° C. withIrbesartan (10 μM), RS504393 (10 μM) or both combined. Cells were thenincubated for a further 30 minutes at 37° C. in the stimulation buffer(10 mM HEPES, pH 7.4, 1 mM CaCl₂, 0.5 mM MgCl₂, 4 mM KCl, 146 mM NaCl,5.5 mM glucose, and 50 mM LiCl) containing 100 nM of AngII, MCP-1 orboth together. The cells were then lysed by adding the HTRF® assayreagents, the Terbium Cryptate-labeled anti-IP1 antibody, and thed2-labeled IP1 analog, previously diluted in the lysis buffer containing1% Triton X-100. The assay was incubated for 1 hour at room temperature,and Terbium Cryptate fluorescence and the time resolved FRET signal weremeasured 50 μs after excitation at 340, 620, and 665 nm, respectively,using the EnVision 2102 multilabel plate reader (PerkinElmer).

Data are normalized as a percentage of AngII-induced IP1 production incells expressing AT1R alone. Data represent mean±SEM of threeindependent experiments performed in triplicate.

As shown in FIG. 6 (top panel), 10 μM of Irbesartan, but not RS504393,substantially abolished the AngII-induced IP1 production in cellsexpressing AT1R-Rluc8. The Irbesartan effect was not substantiallyaltered by its combination with RS504393 in the absence of CCR2co-expression, demonstrating the specificity of the antagonist.

In cells co-expressing both AT1R-Rluc8 and CCR2, in addition toAngII-mediated IP1 response, MCP-1 also seems to stimulate a partial IP1response (FIG. 6: bottom panel). Interestingly, Irbesartan partiallyreduced MCP-1-induced IP1 production, as well as substantiallyinhibiting the response induced by AngII (FIG. 6: bottom panel). Incontrast, RS504393 had little effect on AngII-induced IP1 production,but substantially and selectively inhibited MCP-1-induced IP1 response(FIG. 6: bottom panel). More interestingly, the combination of bothantagonists substantially abolished the IP1 production promoted by bothMCP-1 and AngII as the two receptors are simultaneously inhibited (FIG.6: bottom panel). Together, these data clearly indicate the specificityof the MCP-1-dependent IP1 response via CCR2 and imply that theactivation of both AT1R and CCR2 are required for such a response.Moreover, these findings provide further in vitro evidence for a greaterinhibition of receptor-mediated cellular response, in this case inositolphosphate production, as a consequence of combined receptor inhibition.

Example 5—Upon Co-Expression of AT1R and CCR2 Via Transient Transfectionof HEK293FT Cells, Combined Inhibition of Both Receptors Blocks ArrestinRecruitment to a Greater Extent than Inhibition of Either ReceptorAlone, as Observed Using the Opposite Orientation of Tagged VersusUntagged Receptor and Using Either of Two Different AngII Antagonists

The combined effect of CCR2 and AT1R inhibition in vitro wasinvestigated by using RS504393 in combination with Irbesartan orEXP3174, the active metabolite of Losartan.

FIG. 7 shows the effect of AT1R and CCR2 blockade on β-arrestin2recruitment. HEK293FT cells were transiently transfected by the plasmidscoding for CCR2-Rluc8 and β-arrestin2-Venus in the absence (top panel)and presence (bottom panel) of haemagglutinin (HA)-tagged AT1R.

Cells were harvested 24 h post-transfection in HEPES-buffered phenolred-free complete medium containing 5% FCS and added to apoly-L-lysine-coated white 96-well plate. 48 h post-transfection, theplate was incubated at 37° C., 5% CO₂ for 2 hours with 30 μM EnduRen(Promega) to ensure substrate equilibrium was reached.

Cells were first pre-incubated or not for 30 minutes at 37° C. withIrbesartan (10 μM), EXP3174 (the active metabolite of Losartan; 10 μM),RS504393 (10 μM) or combinations of Irbesartan and RS504393 or EXP3174and RS504393. Then cells were stimulated or not for 30 minutes at 37° C.with 100 nM of AngII, MCP-1 or both together and the BRET signal wasmeasured.

BRET detection was carried out in live cells by measuring sequentiallight emissions at 400-475 nm and 520-540 nm before and after agonistaddition. The BRET signal was calculated by subtracting the ratio of520-540 nm emission over 400-475 nm emission for a vehicle-treated cellsample from the same ratio for a second aliquot of the same cellstreated with ligand (ligand-induced BRET).

As shown in FIG. 7 (top panel), in cells co-expressing CCR2-Rluc8 andβ-arrestin2-Venus, 100 nM of AngII had no effect. 10 μM of RS504393, butneither Irbesartan nor EXP3174, substantially blocked the MCP-1-inducedBRET response and their combination did not give any different effect asexpected (FIG. 7: top panel).

However, in cells co-expressing CCR2-Rluc8, β-arrestin2-Venus andHA-tagged AT1R where both AngII and MCP-1 induced BRET increases todifferent degrees, RS504393 substantially inhibited the MCP-1-inducedbut not the AngII-induced BRET (FIG. 7: bottom panel). Similarly,Irbesartan or EXP3174 partially blocked the AngII- but not theMCP-1-promoted BRET signal (FIG. 7: bottom panel). Notably however, withcombined treatment with MCP-1 and AngII, a substantial BRET signalremained despite treatment with RS504393. Importantly, the combinationof Irbesartan or EXP3174 with RS504393 reduced the BRET response tolevels below that observed with either individual antagonist alone,providing in vitro evidence for a greater inhibition ofreceptor-mediated cellular response, in this case β-arrestinrecruitment, as a consequence of combined receptor inhibition.

Example 6—Upon Co-Expression of AT1R and CCR2 Via Transient Transfectionof HEK293FT Cells, Combined Inhibition of Both Receptors Blocks InositolPhosphate Signalling to a Greater Extent than Inhibition of EitherReceptor Alone, as Demonstrated with Another AngII Antagonist, EXP3174,the Active Metabolite of Losartan

RS504393 was used in combination with EXP3174 to investigate thecombined effect of CCR2 and AT1R inhibition in vitro.

FIG. 8 shows the effect of AT1R and CCR2 blockade on inositol phosphateproduction. HEK293FT cells were transiently transfected with theplasmids coding for AT1R-Rluc8 and β-arrestin2-Venus in the absence (toppanel) and presence (bottom panel) of untagged CCR2. 48 hpost-transfection, cells were used to generate the agonist-inducedinositol (1) phosphate (IP1) production measurements using the IP-One Tbkit (Cisbio Bioassays, Bagnol sur Ceze, France).

Cells were first pre-incubated or not for 30 minutes at 37° C. withEXP3174 (10 μM), RS504393 (10 μM) or both combined. Cells were thenincubated for a further 30 minutes at 37° C. in the stimulation buffer(10 mM HEPES, pH 7.4, 1 mM CaCl₂, 0.5 mM MgCl₂, 4 mM KCl, 146 mM NaCl,5.5 mM glucose, and 50 mM LiCl) containing 100 nM of AngII, MCP-1 orboth together. The cells were then lysed by adding the HTRF® assayreagents, the Terbium Cryptate-labeled anti-IP1 antibody, and thed2-labeled IP1 analog, previously diluted in the lysis buffer containing1% Triton X-100. The assay was incubated for 1 hour at room temperature,and Terbium Cryptate fluorescence and the time resolved FRET signal weremeasured 50 μs after excitation at 340, 620, and 665 nm, respectively,using the EnVision 2102 multilabel plate reader (PerkinElmer). Data areshown as induced IP1 (arbitrary units). The IP-One Tb kit is acompetition assay and so induction of IP1 results in a decrease inabsolute assay signal. Therefore, the induced IP1 (arbitrary units) isgenerated by subtracting the ligand-induced assay signal from the basalassay signal.

As shown in FIG. 8 (top panel), 10 μM of EXP3174, but not RS504393,abolished the AngII-induced IP1 production in cells expressingAT1R-Rluc8. The EXP3174 effect was not substantially altered by itscombination with RS504393 in the absence of CCR2 co-expression,demonstrating the specificity of the antagonist.

In cells co-expressing both AT1R-Rluc8 and CCR2, in addition toAngII-mediated IP1 response, MCP-1 also seems to stimulate a partial IP1response (FIG. 8: bottom panel). EXP3174 substantially inhibited theresponse induced by AngII (FIG. 8: bottom panel). In contrast, RS504393had little effect on AngII-induced IP1 production, but substantially andselectively inhibited MCP-1-induced IP1 response (FIG. 8: bottom panel).More interestingly, the combination of both antagonists substantiallyabolished the IP1 production promoted by both MCP-1 and AngII as the tworeceptors are simultaneously inhibited (FIG. 8: bottom panel). Thesefindings provide further in vitro evidence for a greater inhibition ofreceptor-mediated cellular response, in this case inositol phosphateproduction, as a consequence of combined receptor inhibition.

Example 7—Specific Activation of the AT1R Inhibits MCP-1-MediatedCoupling to Gαi1 in a Dose-Dependent Manner, Providing In Vitro Evidencefor AT1R Modulating CCR2 Function and Providing Further Rationale forInhibition of CCR2 in Addition to AngII

FIG. 9 shows dose-response curves indicating the effect of activatingCCR2 in the absence and presence of activated AT1R in terms of Gαi1coupling as measured by Ligand-induced BRET. HEK293FT cells weretransiently transfected by the plasmids coding for Gα_(i1)-Rluc8 andCCR2-YFP in the absence (top panel) and presence (bottom panel) ofhaemagglutinin (HA)-tagged AT1R. 48 h post-transfection, cells were usedto generate the agonist-induced BRET signal data in live cells atvarious concentrations of MCP-1 or at various concentrations of AngII inthe presence of 100 nM MCP-1.

Upon addition of BRET substrate, BRET detection was carried out in livecells by measuring sequential light emissions at 400-475 nm and 520-540nm before and after agonist addition. The BRET signal was calculated bysubtracting the ratio of 520-540 nm emission over 400-475 nm emissionfor a vehicle-treated cell sample from the same ratio for a secondaliquot of the same cells treated with ligand (ligand-induced BRET).Activation of CCR2 resulted in a decrease in BRET ratio, indicating thatactivation resulted in a change of conformation of a Gαi1 proteinpre-assembled with CCR2, as has been recently described for thePAR1-Gαi1 interaction (Ayoub M A, Trinquet E, Pfleger K D G and Pin J P(2010) Differential association modes of the thrombin receptor PAR1 withGαi1, Gα12 and β-arrestin 1. FASEB J 24: 3522-3535). Therefore, the datahave been presented as ‘Change in BRET (% of Control)’ such that thechange in BRET signal observed with 1 μM MCP-1 is designated 100%, andno change in BRET signal being designated 0%.

MCP-1 alters the distance and/or orientation between Rluc8 and YFP fusedto Gαi1 and CCR2 respectively in a dose-dependent manner indicative ofreceptor activation and coupling to Gαi1-mediated signalling. In theabsence of AT1R co-expression, increasing doses of AngII does not alterthe MCP-1-induced change in BRET signal observed (FIG. 9: top panel). Incontrast, when AT1R is co-expressed, AngII inhibits the MCP-1-inducedchange in BRET signal in a dose-dependent manner (FIG. 9: bottom panel).

This example provides evidence for AngII inhibiting the MCP-1-inducedactivation of Gαi1 coupling by CCR2. Therefore, blockade of AngII aloneusing an AngII antagonist, would be expected to remove this inhibitionof MCP-1-induced Gαi1 signalling. Treatment with a CCR2 pathwayinhibitor in combination with an AngII antagonist would be expected toprevent activation of CCR2-mediated Gαi1 being exacerbated by AT1Rblockade.

Therefore this example provides further in vitro evidence supporting therationale for using a combination of CCR2 pathway inhibitor and AT1Rantagonist.

1-54. (canceled)
 55. A method for the treatment or amelioration of akidney disease associated with proteinuria in a patient being treatedwith at least one angiotensin 1 receptor (AT₁R) blocker or apharmaceutically acceptable salt thereof, the method comprising the stepof further administering to the patient at least one chemokine receptor2 (CCR2) inhibitor or a pharmaceutically acceptable salt thereof,wherein the at least one CCR2 inhibitor comprises a direct CCR2antagonist; an inverse CCR2 agonist; or a negative allosteric CCR2modulator.
 56. The method of treatment or amelioration of claim 1,wherein the AT₁R blocker: a) inhibits or partially inhibits theactivation of AT₁R; b) is selected from the group consisting of: (i) anAT₁R antagonist; (ii) an inverse AT₁R agonist; and (iii) a negativeallosteric AT₁R modulator; and/or c) is selected from the groupconsisting of: irbesartan, olmesartan, eprosartan; losartan; valsartan;telmisartan; candesartan; and ZD-7115.
 57. The method of treatment oramelioration of claim 1, wherein the CCR2 inhibitor: a) inhibits orpartially inhibits CCR2; b) stabilizes the complexes CCR2/CD65,CCR2/CD59 and/or CCR2/CD 16; c) inhibits MCP-1 induced migration andactivation of monocytes and chemotactic migration through the targetingof one or more glycosylphosphatidylinositol (GPI)-anchored proteinsselected from the group consisting of CD55, CD59 and CD16; and/or d) isselected from the group consisting of: propagermanium; RS504393;MLN-1202; 1NCB8696; MK-0812; CCX140; PF-4136309; RS102895; andBMS-741672.
 58. The method of treatment or amelioration of claim 1,wherein the kidney disease associated with proteinuria is selected fromthe group comprising: fibrotic disorders in the kidney, chronic kidneydisease caused by diabetic nephropathy, renal insufficiency, renalfailure conditions, diabetic nephropathy, glomerulonephritis, glomerularsclerosis, proteinuria of primary renal disease.
 59. The method oftreatment or amelioration of claim 1, wherein the patient is beingtreated with about 5 mg to 1 g of the AT₁R blocker or a pharmaceuticallyacceptable salt thereof, and is administered about 5 mg to 1 g of theCCR2 inhibitor or a pharmaceutically acceptable salt thereof.